Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

Abstract

The present study examines the effect of reversed-phase high-performance liquid chromatography (RP-HPLC) column diameter (1mm to 9.4mm I.D.) on the one-step slow gradient preparative purification of a 26-residue synthetic antimicrobial peptide. When taken together, the semi-preparative column (9.4mm I.D.) provided the highest yields of purified product (an average of 90.7% recovery from hydrophilic and hydrophobic impurities) over a wide range of sample load (0.75-200mg). Columns with smaller diameters, such as narrowbore columns (150x2.1mm I.D.) and microbore columns (150x1.0mm I.D.), can be employed to purify peptides with reasonable recovery of purified product but the range of the crude peptide that can be applied to the column is limited. In addition, the smaller diameter columns require more extensive fraction analysis to locate the fractions of pure product than the larger diameter column with the same load. Our results show the excellent potential of the one-step slow gradient preparative protocol as a universal method for purification of synthetic peptides.

Fig. 1

Sequence of peptide D-V13K_D (top) and representation as a helical net (left) and helical wheel (right). In the helical net, non-polar residues making up the non-polar face of the amphipathic peptide are circled. In the helical wheel, the non-polar face of the amphipathic peptide is indicated by an open arc, whilst the polar face is indicated by a solid arc. The lysine residue at position 13 of the sequence is denoted by a triangle on the non-polar face of both representations. Ac denotes N^α-acetyl and amide denotes C^α-amide. One-letter codes are used for the amino acid residues.

Fig. 2

Analytical RP-HPLC profile of the crude peptide. Column: narrowbore Zorbax 300SB-C₈ column (150 × 2.1 mm ID; 5 μm particle size, 300 Å pore size). Conditions: linear AB gradient (1% acetonitrile/min) at a flow-rate of 0.25 ml/min at room temperature, where eluent A is 0.2% aq. TFA and eluent B is 0.2% TFA in acetonitrile. Panel B represents a four-fold magnification of Panel A. P denotes the desired product.

Fig. 3

Analytical RP-HPLC profiles of the pooled hydrophilic impurities, product and hydrophobic impurities following purification on a semi-preparative reversed-phase column. Column and conditions: same as Fig. 2. Panels A, B and C show analytical profiles of the pooled hydrophilic impurities, product and hydrophobic impurities, respectively, obtained following purification of a 200 mg sample load of crude peptide dissolved in 20 ml 0.2% aq. TFA. Panels D, E and F show analytical profiles of the pooled hydrophilic impurities, product and hydrophobic impurities, respectively, obtained following purification of a 6 mg sample load of crude peptide dissolved in 0.6 ml 0.2% aq. TFA. Panels A, C, D and F represent three-fold magnifications compared to panels B and E. P denotes the desired product.

Fig. 4

Analytical RP-HPLC of fractions obtained during purification of 100 mg (top) and 200 mg (bottom) of crude peptide. Column and conditions: same as Fig. 2. P denotes the desired product and I denotes impurities.

Fig. 5

Comparison of the preparative RP-HPLC profiles of 0.75 mg crude peptide on semi-preparative (Panel A) and microbore (Panel B) columns. In Panel B, fraction containing pure product is denoted as a black bar. Product fraction numbers indicate 0.5 min per fraction.

Fig. 6

Recovery of purified product obtained following purification of varying amounts of crude peptide on columns with different diameters. Sample loads are indicated at the top of each plot. Data obtained from Table 1.